1. Field of the Invention
The present invention relates to the prevention of vascular restenosis either through drug therapy or by gene therapy.
2. Description of Related Art
Thrombin, the central enzyme of coagulation cascade, elicits a number of cellular responses relevant to haemostasis, fibrinolysis, cell growth, inflammation, wound healing and tissue remodeling. Thrombin-specific inhibitors, hirudin and hirulog-1, reduced ischemic events in coronary artery disease (CAD) patients.
Restenosis is one of the major concerns for the treatment of CAD using therapeutic vascular interventions. Classical anti-coagulants did not effectively prevent restenosis but increased the frequency of bleeding complications. Thrombin inhibitors have been considered as potential candidate for the treatment of restenosis, although long-term benefit of the thrombin inhibitors on restenosis has not been shown in humans.
Recent studies in experimental animal models have demonstrated that the regimen for thrombin inhibitors is an important determinant of their therapeutic efficacy. The major limitation for using prolonged and large doses of thrombin inhibitors in patients is bleeding complications. At effective doses, hirulog-1 increased bleeding tendency in rats.
Thrombin, a key enzyme for haemostasis and several other important physiological processes, is a product of activated coagulation cascade. (Fenton, et al 1998). The generation of thrombin takes place on phospholipid-rich cell surfaces via a series of proteolytic reactions. Surface of activated platelets or injured vascular intima provides an optimal locus for coagulation reactions which stimulate the generation of thrombin from its precursor. Thrombin is involved at all levels of haemostasis, including plasma, blood cells and vasculature. Thrombin stimulates the formation of fibrin clot. It also activates several other coagulation factors (factor V, VIII and XIII) which further increase the generation of thrombin. Thrombin is a potent agonist for platelet secretion and aggregation. The secretory products of platelets enhance coagulation and thrombin formation. On the other hand, thrombin activates protein C by increasing its binding to thrombomodulin on endothelial cell (EC) surface (Esmon C T. 1983). Activated protein C inhibits coagulation by inactivating factor Va and VIIIa.
Besides its central role in hemostasis, thrombin stimulates the production of plasminogen activators (PA) and their major physiological inhibitor, plasminogen activator inhibitor-1 (PAI-1) in vascular EC and smooth muscle cells (SMC). Those fibrinolytic regulators modulate the generation of plasmin, a serine proteinase functions in dissolving fibrin clots and tissue remodeling (appendix IV). Thrombin induces the secretion of α-granule contents from platelets, including P-selectin (CD62P), an adhesion molecule involved in platelet aggregation, inflammation and thrombosis (Nguyen A, Gemmell et al, 1998). Thrombin is also a potent mitogen. It stimulates the expression of multiple growth factors in vascular cells, including platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), heparin binding epithelial growth factor (HBEGF) and transforming growth factor-β (TGF-β). Elevated expression of those growth factors has been detected in injured arterial walls (Ross R, et al 1986; Stouffer G A et al, 1998). Cellular responses of thrombin are usually mediated through the activation of its membrane receptor (Huang R. et al 1991).
The thrombin receptor has a long extracellular extension, which contains a proposed cleavage site for the enzyme and a binding site for thrombin. The proteolytic cleavage of thrombin receptor generates a short peptide with a newly exposed NH2 terminus, known as “tethered ligand”, which helps to activate the receptor. The thrombin receptor has seven hydrophobic segments spanning the lipid bilayer of plasma membrane and its intracellular extension is coupled with G protein (Rasmussen U B et al 1991; Vu T K et al 1991).
Inhibitory G protein-coupled thrombin receptor, tyrosine kinase, phospholipase C and protein kinase C are involved in the regulation of thrombin-induced PAI-1 production in vascular SMC (Shen G X et al 1998; Ren S et al 1997). Thrombin may elicit multiple cellular responses related to cell growth and tissue remodeling via transmembrane signaling, which may be blocked by inhibitors targeted to the thrombin receptor or corresponding signaling pathway.
Vascular procedures, including angioplasty and endarterectomy, relieve atherosclerotic vascular stenosis without surgical intervention (Ryan T J et al 1988; Lincoff A M et al, 1993). However, angiographic or clinical restenosis occurred in 30%-50% of patients within 6 months after vascular procedures (Libby P et al, 1992; Clowes A W, Reidy M A, 1991). Histological studies demonstrated that SMC are the major cellular component in injury-induced neointima and restenotic lesions (Narins et al, 1998). Multiple biological processes are involved in the development of restenosis, including platelet activation, thrombus formation, SMC proliferation, tissue remodeling, EC activation, inflammation, oxidation and the expression of growth factors and oncogens (Scwartz S M et al, 1996). Proteases, including plasmin and metalloproteinases, may contribute to tissue remodeling and neointima formation following vascular injury (Libby P et al, 1992; Popma J J et al, 1993). The formation of plasmin is activated by tissue and urokinase plasminogen activators (tPA and uPA). Transgenic mice expressing uPA developed less extent of neointima induced by balloon injury (Carmeleit P, et al 1997). Increased levels of PAI-1 were found in plasma of patients developed angioplasty-induced restenosis (Ishiwata S et al 1997). The generation of uPA, tPA and PAI-1 from vascular cells is stimulated by thrombin. Increased levels of P-selectin, a marker for platelet aggregation responding to thrombin stimulation, were detected in plasma of patients with post-angioplasty restenosis (Ischinger T A, 1998; Ishiwata S et al, 1997, Tsakirirs D A et al, 1999). Stent implantation following angiography has virtually abolished periprocedural obstructive dissection and delays the occurrence of restenosis. The rates of intracoronary thrombosis and late restenosis were not reduced by stent implantation. Recurrent in-stent restenosis or vascular complications frequently occurred in the receivers. Stent-related vascular complications were found in some receivers. Extensive anticoagulant treatment is essential for post-stent management, which greatly increases the risk of bleeding (Safian R D et al, 1990; Carmeliet P et al 1997). Vascular restenosis remains as one of the major concerns for the treatment of atherosclerotic cardiovascular diseases using therapeutic vascular interventions.
Pharmacological prevention of vascular restenosis is highly demanded. Traditional anti-thrombotic agents, heparin, aspirin or oral anti-coagulants, did not effectively reduce the frequency of restenosis and increased bleeding complications (Buchanan M R et al, 1999). A variety of drugs has been tested in experimental animal vascular injury models and some of them were used in clinical trials (Lefkovitis J et al, 1997; Liu L Y et al, 1996). Several types of drugs appear to be potential, including PDGF antagonist (trapidil) (Maresta A, 1994), antioxidants (probucol, vitamin C and E) (Ferns G A et al 1992; Vermylen J, 1995) and thrombin-specific inhibitors (hirudin, hirulog-1) (Sarembock I J et al 1996; Gallo R et al 1998). The efficacy and safety of all those drugs for the prevention of restenosis in humans remains to be determined. Chimeric monoclonal antibody for glycoprotein IIB/IIIA receptor (abciximab or Reopro) improved the outcome of post-angioplasty CAD patients (Lefkovits J, 1996) but did not reduce intima hyperplasia or restenosis (Deitch J S et al, 1998; McGregor M et al, 1999).
Anti-thrombin III inhibits clotting factor by forming complexes with them. Heparin enhances the effect of anti-thrombin III by thousands-fold and it is the most commonly used anticoagulant in clinical practice (Hull R D, 1992). The major complication of heparin treatment is haemorrhage. Most of bleeding complications occurred at the puncture sites, but around 0.9% of heparin receivers developed intracranial haemorrhage (J Neurol Neurosurg Psychiatry 1996). In some individuals, heparin treatment caused thrombocytopenia and intravascular thrombosis. Heparin is not effective for thrombolysis, which probably is due to lack of access to thrombin in clots (Weitz J I et al, 1990). Besides, heparin suppressed the production of tPA but not PAI-1 in cultured SMC, which potentially attenuates fibrinolytic activity (Au Y P T et al, 1991). Low molecular weight (LMW) heparin caused less profound effect on haemorrhagic complications than regular heparin (Hirsh J et al., 1999). However, treatments with LMW heparin did not significantly reduce angiographic or clinical restenosis (Gimple L W et al, 1999).
Thrombolytic agents, including streptokinase, recombinant tPA, uPA and anisoylated plasminogen streptokinase activator complex, have been approved for the treatment of acute myocardial infarction. Those agents were also effective in relieving peripheral vascular thrombosis, pulmonary embolism and the restoration of the patency of catheter. Thrombolytic agents directly stimulate the formation of plasmin which functions in dissolving fibrin clots. The activity of tPA is greatly increased in the presence of fibrin (Garabedian H D et al, 1987). The major concern for using thrombolytic agents is high incidence of life-threatening bleeding complications and reocclusion. Intracranial haemorrhage, occurred in 0.5% of the receivers. Reocclusion of vessels following thrombolytic treatment was found in 10%-20% of receivers due to unidentified mechanism (Lavie C J et al 1990). Excess expression of uPA, promoted neointima formation in arteries following vascular injury in animal models (Carmeleit P et al, 1997). Those findings imply that thrombolytic agents and heparin may not be ideal candidates for preventing restenosis.
Hirudin is a 65 amino acid protein originally isolated from medicinal leech (Harvery R P et al 1986). It is the most potent natural thrombin-specific inhibitor. Results of the Helvetica trial showed that hirudin treatment reduced early cardiac events but had no long-term benefit for restenosis in post-angioplasty patients. In that study, hirudin was given in bolus injection plus intravenous infusion for 24 hours with and without subcutaneous booster injections. No description was provided from that report on the starting time of hirudin infusion relevant to angioplasty. Similar frequency of major bleeding (6%-7%) was found in hirudin receivers compared to conventional heparin treatment (Serruys R W, 1995). More recent studies indicated that modifications on the regimen of hirudin may considerably improve its anti-restenosis effect in animals. Prolonged infusion of hirudin for two weeks effectively reduced angioplasty-induced stenosis in swine model compared to bolus or short infusion (Gallo, R, et al, 1998). Infusion of hirudin for 24 hours started before angioplasty significantly reduced angioplasty-induced restenosis in atherosclerotic rabbits compared to four hours or delayed infusion of hirudin (Thome L M et al, 1998). Intravascular gene transfer of hirudin reduced balloon catheter injury-induced neointima formation by 50% in rats (Rade, J J, et al, 1996). Those findings suggest that the earlier results from clinical trials on the ineffectiveness of hirudin on restenosis may need to be re-evaluated using rationalized regimen. Results from phase II clinical trials suggested that hirudin reduced ischemic events in angina, post-myocardial infarction or post-angioplasty patients without significant increase in bleeding complications compared to conventional heparin treatment (Van den Bos, et al, 1993; Topol E J, et al, 1994; Cannon, C P, et al 1994; Lee, L V, 1995). Phase III clinical trials in large scales of patients indicated that hirudin caused high incidence of life-threatening haemorrhage (Adgey, A A, 1996). One of the trials found that the incidence of major bleeding complications at non-cranial sites in hirudin-treated patients (7%) was over two-fold higher than conventional heparin treatment (3%), and the trial was prematurely suspended (Antman, E M, 1996). Two other trials were stopped after the enrollment of 302 and 2,564 patients due to high incidence of intracranial haemorrhage in hirudin-treated patients compared to conventional heparin treatment (GUSTO, 1994; Neuhaus, K L, et al, 1994). High frequencies of life-threaten bleeding complications limit the application of hirudin in stable CAD patient.
Hirulogs are a group of synthetic thrombin-specific inhibitors which conserve the major active sites of hirudin. Hirulog-1, a 20-residue peptide, is the strongest hirulog (Maraganore, J M, 1990; Ofosu, F A, 1992). It is composed of a N-terminal domain (D-FPRP), which blocks the enzymatic active site, and a C-terminal domain (NGDFEEIPEYL SEQ. ID NO:3) inhibiting the binding of thrombin to its receptor. Hirulog-1 treatment effectively prevented acute cardiovascular events in post-myocardial infarction (Lidon, R M, 1993), coronary angiography (Lidon, R M, 1994) and percutenous transluminal coronary angioplasty (PTCA). (Bittl, J A, 1995). Since hirulog-1 is a peptide in nature and is quickly metabolized in gastrointestinal tract via oral intake, it has only been administrated via intravenous route as other peptide drugs. The plasma half-life of hirulog-1 in human is 15-20 minutes (Fenton, J W, 1992). Allergy to hirulog-1 has not been reported probably due to the weak antigenicity of the peptide. Phase III clinical trials demonstrated that hirulog-1 increased bleeding complications in CAD patients following angioplasty, but the incidence was lower than conventional heparin treatment (Bittl, J A, 1995). Continuous intravenous infusion of hirulog-1 reduced platelet deposition 30 minutes after endarterectomy with and without aspirin administration (Hamelink, J K, 1995; Jackson, M R et al, 1996). Hirulog-1 inhibits thrombin-induced production of PAI-1 in cultured arterial SMC (appendix III). In diet-induced atherosclerotic rabbits, hirulog-1 infusion reduced restenosis induced by angioplasty compared to heparin treatment (Sarembock, I J, et al, 1996). Hirulog-1-impregnated silicone polymers placed around adventitia surface of stented segments did not inhibit stent-induced stenosis in pig carotid artery. The results of that study was questioned by the uncertainty of the delivery of hirulog-1 to vascular lumen (Muller, D W, 1996). The applicant's group demonstrated that hirulog-1 inhibited platelet deposition on intima denuded by balloon catheter in rats (Shen, G, et al, 1997). Multiple prolonged infusions of hirulog-1 (1 mg/kg/hours for 4 hours for 6 times, immediately following injury and every other days after for 5-times) inhibited balloon catheter injury-induced increase in neointima/media ratio by 50% in rat carotid arteries. Some results demonstrated that hirulog-1 infusions attenuated the abundance of PDGF in neointima of rat carotid arteries. Tail bleeding time and aPTT were significantly elongated by hirulog-1 treatment in rats. Those results indicated that hirulog-1 effectively reduces balloon catheter injury-induced neointima formation in rats. The preventive effect of hirulog-1 on neointima formation may result, at least partially, from its inhibition on PDGF expression in vascular wall. Impaired coagulation activity and prolonged bleeding time were detected in hirulog-1-treated rats, which potentially cause haemorrhage when large doses of the inhibitor are administrated.
As discussed, Hirulog-1, a synthetic thrombin inhibitor, is effective in preventing ischemic events in coronary artery disease and causes significantly less bleeding complications than classical anticoagulants. Studies have demonstrated that hirulog-1 inhibited thrombin-induced PAI-1 production in SMC (Ren et al J Vas Res 1997;29:337-42). Hirulog-1 inhibited thrombin-induced SMC proliferation (Ren et al unpublished observations). Bolus injection of hirulog-1 transiently reduced platelet deposition on intima following balloon catheter injury in rats. Multiple prolonged intravenous infusions of hirulog-1 partially prevented balloon injury-induced neointima formation.
Clinical studies demonstrate that hirulog-1 treatment was equal or more effective on preventing ischemic events in coronary artery disease and caused significantly less major bleeding complications than classical anticoagulants. Recent studies demonstrated that hirulog-1 inhibited the production of plasminogen activator inhibitor-1 and thymidine incorporation in cultured smooth muscle cells (SMC). In addition, hirulog-1 injection inhibited balloon injury-induced platelet deposition, thrombin activation and reduced neointima formation in carotid arteries in rats. Hirulog-1 contains unnatural amino acid which prevents this peptide from being expressed in mammalian cells.
Vascular restenosis induced by angioplasty and other vascular intervention is a major concern for the treatment for coronary heart disease. Ischemic events occur within in months of 40% patients receiving angioplasty. No effective treatment is available for vascular restenosis. It would therefore be useful to develop a treatment of vascular restenosis without any of the drawbacks set forth above.